Transcription profiling of mouse ovaries from FORKO mutants animals

To understand consequences of loss of FSH receptor, we used genetically altered mouse mutants with deletion of this receptor. Follitropin Receptor Knockout (FORKO) mice present various developmental and age-dependent abnormalities such as infertility, senescence and increased tumor incidences in addition to other phenotypes. To explore why the atrophic ovaries of FORKO mice develop ovarian anomalies later in life, we used expression profiling studies to gain a more comprehensive view of genes that are misregulated. Experiment Overall Design: Mice were housed under controlled temperature and constant light (12 h of light, 12 h of darkness), with food and water provided ad libitum. The female mice used in this experiment were derived by breeding heterozygotes of the 129T2 svEmsJ background. Experiment Overall Design: For RNA extraction, both the ovaries from 8 month old mice were carefully dissected and pooled. Tissues were frozen in liquid nitrogen and homogenized in TRIZOL (Invitrogen, Burlington, Ontario) and processed in accordance with the manufacturer's protocol. Experiment Overall Design: Affymetrix 39 K GeneChip Mouse Genome 430 2.0 Array has over 45000 probe sets which analyses 39000+ transcripts and variants from over 34000 well characterized mouse genes. A total of 1 µg of double-stranded cDNA was transcribed in vitro using the Bioarray High-Yield RNA Transcript Labeling Kit (Enzo, Farmingdale, NY) according to the manufacturer's instructions using biotinylated CTP and UTP (Enzo, Farmingdale, NY). Following 5-hr incubation at 37°C, the resultant biotin-labeled cRNA was purified with RNAeasy columns (Qiagen) and eluted in 40 µl of RNAse-free water. The concentration of biotin-labeled cRNA was determined by RNA bio-analyzer. Target cRNAs corresponding to either wild type ovary or FORKO ovarian tissue were hybridized to an individual GeneChip from an identical lot of Affymetrix 39 K GeneChip Mouse Genome 430 2.0 array for 16 hr. GeneChip arrays were washed and stained using antibody-mediated signal amplification and the Affymetrix Fluidics Station's standard Eukaryotic GE Wash 2 protocol, using Affymetrix equipment and protocols (Affymetrix, Santa Clara, CA).为明确促卵泡激素受体（FSH receptor）缺失所产生的生物学效应，本研究采用经基因编辑构建的该受体敲除小鼠模型。促卵泡激素受体敲除（Follitropin Receptor Knockout, FORKO）小鼠可出现多种发育相关及年龄依赖性异常，除其他表型外，还包括不育、衰老进程加速及肿瘤发生率升高等特征。为探究FORKO小鼠的萎缩卵巢为何在生命后期出现卵巢异常，本研究通过表达谱分析技术，全面解析发生表达失调的基因全貌。 实验整体设计：所有小鼠均饲养于环境可控的温控与光照条件下（12小时光照、12小时黑暗循环），自由进食饮水。本实验所用雌性小鼠均由129T2 svEmsJ背景的杂合子小鼠交配繁育获得。 实验整体设计：用于RNA提取的卵巢组织均取自8月龄小鼠，经精细解剖后混合收集。组织样品经液氮速冻后，在TRIZOL（Invitrogen, Burlington, Ontario）中完成匀浆，并严格按照制造商提供的实验流程进行后续处理。 实验整体设计：本实验采用Affymetrix 39 K GeneChip小鼠基因组430 2.0芯片，该芯片包含超过45000个探针组，可对34000余个已充分注释的小鼠基因对应的39000余种转录本及变异体进行检测。取1 μg双链cDNA，采用Bioarray High-Yield RNA Transcript Labeling Kit（Enzo, Farmingdale, NY），按照制造商说明书，以生物素标记的CTP和UTP进行体外转录。于37℃孵育5小时后，所得生物素标记cRNA经RNAeasy层析柱（Qiagen）纯化，并用40 μl无RNA酶水洗脱。通过RNA生物分析仪测定生物素标记cRNA的浓度。将野生型卵巢或FORKO卵巢组织对应的靶标cRNA，与同一批次的Affymetrix 39 K GeneChip小鼠基因组430 2.0芯片进行16小时杂交。随后采用抗体介导的信号放大技术，按照Affymetrix Fluidics Station标准真核生物GE清洗2号流程，使用Affymetrix配套设备与实验方案（Affymetrix, Santa Clara, CA）完成基因芯片的洗涤与染色。